Who discovered malaria?

A French Army physician Charles Louis Alphonse Laveran discovered that malaria was caused by microscopic parasite (now called "Plasmodium falciparum") in 1880. But scientists were sceptical until Golgi intervened. It was Golgi who helped him prove that malarial parasite was a microscopic protozoan. From 1885, Golgi studied the malarial parasite and its transmission. He established two types of malaria, tertian and quartan fevers caused by "Plasmodium vivax" and "Plasmodium malariae" respectively. In 1886, he discovered that malarial fever (paroxysm) was produced by the asexual stage in the human blood (called erythocytic cycle, or Golgi cycle). In 1889-1890, Golgi and Ettore Marchiafava described the differences between benign tertian malaria and malignant tertian malaria (the latter caused by "P. falciparum"). By 1898, along with Giovanni Battista Grassi, Amico Bignami, Giuseppe Bastianelli, Angelo Celli and Marchiafava, he confirmed that malaria was transmitted by "Anopheline" mosquito.

Camillo Golgi

Camillo Golgi (; 7 July 1843 – 21 January 1926) was an Italian biologist and pathologist known for his works on the central nervous system. He studied medicine at the University of Pavia (where he later spent most of his professional career) between 1860 and 1868 under the tutelage of Cesare Lombroso. Inspired by pathologist Giulio Bizzozero, he pursued research in nervous system. His discovery of a staining technique called black reaction (sometimes called Golgi's method or Golgi's staining in his honour) in 1873 was a major breakthrough in neuroscience. Several structures and phenomena in anatomy and physiology are named for him, including the Golgi apparatus, the Golgi tendon organ and the Golgi tendon reflex. He is recognized as the greatest neuroscientist and biologist of his time.

Golgi and the Spanish biologist Santiago Ramón y Cajal were jointly given the Nobel Prize in Physiology or Medicine 1906 "in recognition of their work on the structure of the nervous system".

Camillo Golgi was born in July 1843 in the village of Corteno, in the province of Brescia (Lombardy), Italy. The village is now named Corteno Golgi in his honour. His father Allessandro Golgi was a physician and district medical officer, originally from Pavia. In 1860, he entered the University of Pavia to study medicine, and earned his medical degree in 1865. He did an internship at the San Matteo Hospital (now IRCCS Policlinico San Matteo Foundation). During his internship he briefly worked as a civil physician in the Italian Army, and as assistant surgeon at the Novara Hospital (now Azienda Ospedaliero Universitaria Maggiore della Carità di Novara). At the same time he was also involved in the medical team for investigating cholera epidemic in villages around Pavia.

In 1867, he resumed his academic study under the supervision of Cesare Lombroso. Lombroso was a renowned scientist in medical psychology such as genius, madness and criminality. Inspired by Lombroso, Golgi wrote a thesis on the etiology of mental disorders, from which he obtained his M.D. in 1868. He became more interested in experimental medicine, and started attending the Institute of General Pathology headed by Giulio Bizzozero. Three years his junior, Bizzozero was an eloquent teacher and experimenter, who specialised in histology of the nervous system and the properties of bone marrow. The most important research publications of Golgi were directly or indirectly influenced by Bizzozero. The two became so close that they lived in the same building; and Golgi later married Bizzozero's niece, Lina Aletti. By 1872, Golgi was an established clinician and histopathologist. He, however, had no opportunity as a tenured professor in Pavia to pursue teaching and research in neurology.

Financial pressure prompted him to join the Hospital of the Chronically Ill (Pio Luogo degli Incurabili) in Abbiategrasso, near Milan, as Chief Medical Officer in 1872. To continue research, he set up a simple laboratory on his own in a refurbished hospital kitchen, and it was there that he started making his most notable discoveries. His major achievement was the development of staining technique for nerve tissue called the black reaction (later the Golgi's method). He published his major works between 1875 and 1885 in the journal "Rivista sperimentale di Freniatria e di medicina legale". In 1875, he joined the faculty of histology at the University of Pavia. In 1879, he was appointed Chair of Anatomy at the University of Siena. But the next year, he returned to the University of Pavia as full Professor of histology. From 1879 he also became Professor of General Pathology as well as Honorary Chief ("Primario ad honorarem") at the San Matteo Hospital. He served as Rector of the University of Pavia twice, first between 1893 and 1896, and second between 1901 and 1909. During the First World War (1914-1917), he directed the military hospital Collegio Borrmeo at Pavia. He retired in 1918 and continued to research in his private laboratory till 1923. He died on 21 January 1926.

Golgi and his wife Lina Aletti had no children, and they adopted Golgi's niece Carolina.

Golgi was irreligious in his later life and became an agnostic atheist. One of his former students attempted an unsuccessful deathbed conversion on him.

Central nervous system was difficult to study during Golgi's time because the cells were hard to identify. The available tissue staining techniques were useless for studying nervous tissue. While working as chief medical officer at the Hospital of the Chronically Ill, he experimented with metal impregnation of nervous tissue, using mainly silver (silver staining). In the early 1873, he discovered a method of staining nervous tissue that would stain a limited number of cells at random in their entirety. He first treated the tissue with potassium dichromate to harden it, and then with silver nitrate. Under microscope, the outline of the neuron became distinct from the surrounding tissue and cells. The silver chromate precipitate, as a reaction product, only selective stains some cellular components randomly, sparing other cell parts. The silver chromate particles create a stark black deposit on the soma (nerve cell body) as well as on the axon and all dendrites, providing an exceedingly clear and well-contrasted picture of neuron against a yellow background. This makes it easier to trace the structure of the nerve cells in the brain for the first time. Since cells are selective stained in black, he called the process "la reazione nera" ("the black reaction"), but today it is called Golgi's method or the Golgi stain. On 16 February 1873, he wrote to his friend Niccolò Manfredi:

His discovery was published in the "Gazzeta Medica Italiani" on 2 August 1873.

In 1871, a German anatomist Joseph von Gerlach postulated that the brain is a complex "protoplasmic network", in the form of a continuous network called the reticulum. Using his black reaction, Golgi could trace various regions of the cerebro-spinal axis, clearly distinguishing the different nervous projections, namely axon from the dendrites. He drew up a new classification of cells on the basis of the structure of their nervous prolongation. He described an extremely dense and intricate network, composed of a web of intertwined branches of axons coming from different cell layers ("diffuse nervous network"). This network structure, which emerges from the axons, is essentially different from that hypothesized by Gerlach. It was the main organ of the central nervous system according to Golgi. Thus, Golgi presented the reticular theory which states that the brain is a single network of nerve fibres, and not of discrete cells. Although Golgi's earlier works between 1873 and 1885 clearly depicted the axonal connections of cerebellar cortex and olfactory bulb as independent of one another, his later works including the Nobel Lecture showed the entire granular layer of the cerebellar cortex occupied by a network of branching and anastomosing nerve processes. This was due to his strong conviction in the reticular theory. Golgi's theory was challenged by Ramón y Cajal, who used the same technique developed by Golgi. According to Ramón y Cajal's neurone theory, the nervous system is but a collection of individual cells, the neurones, which are interconnected to form a network.

In addition to this, Golgi was the first to give clear descriptions of the structure of the cerebellum, hippocampus, spinal cord, olfactory lobe, as well as striatal and cortical lesions in a case of chorea. In 1878, he also discovered a receptor organ that senses changes in muscle tension, and is now known as Golgi tendon organ or Golgi receptor; and Golgi-Mazzoni corpuscles (pressure transductors). He further developed a stain specific for myelin (a specialised portion of axon) using potassium dichromate and mercuric chloride. Using this he discovered the myelin annular apparatus, often called the horny funnel of Golgi-Rezzonico.

Golgi studied kidney function during 1882 to 1889. In 1882, he published his observations on the mechanism of renal hypertrophy, which he understood to be due to renal cell proliferation. In 1884, he described tubular cell mitoses in the kidney of a person suffering from tubulointerstitial nephritis, and he noted that the process was an essential part of repairing the kidney tissue. He was the first to dissect out intact nephrons, and show that the distal tubulus (loop of Henle) of the nephron returns to its originating glomerulus, a finding that he published in 1889 ("Annotazioni intorno all'Istologia dei reni dell'uomo e di altri mammifieri e sull'istogenesi dei canalicoli oriniferi". "Rendiconti R. Acad. Lincei" 5: 545–557, 1889).

An organelle in eukaryotice cells now known as Golgi apparatus or Golgi complex, or sometimes simply as Golgi, was discovered by Camillo Golgi. Golgi modified his black reaction using osmium dichromate solution with which he stained the nerve cells (Purkinje cells) of the cerebellum of an owl. He noticed thread-like networks inside the cells and named them "apparato reticolare interno" (internal reticular apparatus). Recognising them to be unique cellular components, he presented his discovery before the Medical-Surgical Society of Pavia in April 1898. After the same was confirmed by his assistant Emilio Veratti, he published it in the "Bollettino della Società medico-chirurgica di Pavia". However, most scientists disputed his discovery as nothing but a staining artefact. Their microscopes were not powerful enough to identify the organelles. By the 1930s, Golgi's description was largely rejected. It was only firmly established after 50 years of its discovery, when electron microscopes were developed.

Golgi, together with Santiago Ramón y Cajal, received the Nobel Prize in Physiology or Medicine in 1906 for his studies of the structure of the nervous system. In 1900 he was named senator by King Umberto I. In 1913 he became foreign member of the Royal Netherlands Academy of Arts and Sciences. He received honorary doctorate from the University of Cambridge, University of Geneva, Kristiania University College, National and Kapodistrian University of Athens, and Paris-Sorbonne University. He was commemorated in stamp by European Community in 1994.

In Pavia several landmarks stand as Golgi's memory.

In 1880, while working in the military hospital in Constantine, Charles Louis Alphonse Laveran discovered that the cause of malaria is a protozoan. He observed the parasites in a blood smear taken from a soldier who had just died of malaria. For this, he received the 1907 Nobel Prize for Physiology or Medicine. This was the first time that protozoa were shown to be a cause of disease. His work helped inspire researchers and veterinarians today to try to find a cure for malaria in animals.

Constantine, Algeria

Constantine ( "", ), also spelled "Qacentina" or "Kasantina", is the capital of Constantine Province in northeastern Algeria. During Roman times it was called Cirta and was renamed "Constantina" in honor of emperor Constantine the Great. It was the capital of the French department of Constantine until 1962. Located somewhat inland, Constantine is about from the Mediterranean coast, on the banks of the tiny Rhumel River (more of a brook).

Constantine is regarded as the capital of eastern Algeria and the commercial center of its region, and it has a population of about 450,000 (938,475 with the agglomeration), making it the third largest city in the country after Algiers and Oran. There are several museums and historical sites located around the city. Constantine is often referred to as the "City of Bridges" due to the numerous picturesque bridges connecting the various hills, valleys, and ravines that the city is built on and around.

Constantine was named the Arab Capital of Culture in 2015.

The city was originally created by the Phoenicians, who called it Sewa (royal city). Later it was renamed Cirta, by the Numidian king Syphax, who turned it into his capital. The city was taken over by Numidia, the country of the Berber people, after the Phoenicians were defeated by Rome in the Third Punic War. In 112 B.C., the city was occupied by Jugurtha, who defeated his half-brother Adherbal. The city later served as the base for Roman generals Quintus Caecilius Metellus Numidicus and Gaius Marius in their war against yugurtha. Later, with the removal of King Juba I and the remaining supporters of Pompey in Africa (c. 46), Julius Caesar gave special rights to the citizens of Cirta, now known as Colonia Sittlanorum.

In 311 AD, during the civil war between emperor Maxentius and usurper Domitius Alexander (a former governor of Africa), the city was destroyed. Rebuilt in 313 AD, it was subsequently named after emperor Constantine the Great, who had defeated Maxentius. Captured by the Vandals in 432, Constantine returned to the Byzantine Exarchate of Africa ("i.e." North Africa) from 534 to 697. It was conquered by the Arabs in the 8th century, receiving the name of "Qacentina".

The city recovered in the 12th century and under Almohad and Hafsid rule it was again a prosperous market, with links to Pisa, Genoa and Venice. After 1529 it was intermittently part of Ottoman Empire, ruled by a Turkish bey (governor) subordinate to the dey of Algiers. Salah Bey, who ruled the city in 1770–1792, greatly embellished it and built much of the Muslim architecture still visible today.
In 1826 the last bey, Ahmed Bey ben Mohamed Chérif, became the new head of state. He led a fierce resistance against French forces, which had invaded Algeria four years later. By 13 October 1837, the territory was captured by France, and from 1848 on until 1962 it was an integral part of the French motherland and centre of the Constantine Département. During the period of French control, Muslim anti-Jewish riots in Constantine in 1934 killed 34 Jews.

During World War II, during the campaign in North Africa (1942–43), Allied forces used Constantine and the nearby cities of Sétif and Bone as operational bases.

During the 11th century, Banu Hilal, an Arab tribe living between Nile and Red Sea, settled in Tunisia, Tripolitania (western Libya) and Constantinois (eastern Algeria) which was Constantine party.

Constantine is situated on a plateau at an elevation above sea level. The city is framed by a deep ravine and has a dramatic appearance. The city is very picturesque with a number of bridges over Rhumel River and a viaduct crossing the ravine. The ravine is crossed by seven bridges, including Sidi M'Cid bridge. Constantine is the railhead of a prosperous and diverse agricultural area. It is also a centre of the grain trade and has flour mills, a tractor factory, and industries producing textiles, wool, linen and leather goods. Algeria and Tunisia serve as its markets.

Constantine has a Mediterranean climate (Köppen climate classification "Csa"), with hot, dry summers and mild, moist winters.

The city is framed by a deep ravine and has a dramatic appearance. In 1911, Baedeker described it as "resembling the Kasba of Algiers, the picturesque charm of which has so far been marred by the construction of but a few new streets."

Nearby are

The topography of the city is unique and it determines the need for bridges. At the end of the 19th century, Guy de Maupassant wrote: "Eight bridges used to cross this ravine. Six of these bridges are in ruins today." Today the most important bridges are:

Constantine has 2 universities: Mentouri Public University, designed by the Brazilian architect Oscar Niemeyer, and Algerian architect Rashid Hassaine, Zerzara maths and architecture university, Emir Abdelkader University which is one of the biggest Islamic universities with many faculties covering religious studies, foreign languages, literature. Constantine's new town "nouvelle ville ali mendjeli" has two big universities: Université Constantine 2 known as "lella nsoumer" offering maths, computer and economy majors, and the new university is actually a university pole with more than 20,000 students, 17 faculties and more than 40,000 residents. It is now the largest African university under the name of "Université de Constantine 3".

Constantine is served by Mohamed Boudiaf International Airport.

Constantine is twinned with:

Constantine has been the hometown of many noteworthy people in Algeria and France.

A German physician, Johann Friedrich Meckel, must have been the first to see "P. falciparum" but not knowing what it was. In 1847 he reported the presence of black pigment granules from the blood and spleen of a patient who died of malaria. The French Army physician Charles Louis Alphonse Laveran, while working at Bône Hospital (now Annaba in Algeria), correctly identified the parasite as a causative pathogen of malaria in 1880. He presented his discovery before the French Academy of Medicine in Paris, and published it in "The Lancet", in 1881. He gave the scientific name "Oscillaria malariae". But his discovery was received with skepticism mainly because by that time leading physicians such as Theodor Albrecht Edwin Klebs and Corrado Tommasi-Crudeli claimed that they had discovered a bacterium (which they called "Bacillus malariae") as the pathogen of malaria. Laveran's discovery was widely accepted only after five years when Camillo Golgi confirmed the parasite using better microscope and staining technique. Laveran was awarded the Nobel Prize in Physiology or Medicine in 1907 for his work. In 1900, the Italian zoologist Giovanni Battista Grassi categorized "Plasmodium" species based on the timing of fever in the patient; malignant tertian malaria was caused by "Laverania malariae" (now "P. falciparum"), benign tertian malaria by "Haemamoeba vivax" (now "P. vivax"), and quartan malaria by "Haemamoeba malariae" (now "P. malariae").

Plasmodium falciparum

Plasmodium falciparum is a unicellular protozoan parasite of humans, and the deadliest species of "Plasmodium" that cause malaria in humans. It is transmitted through the bite of a female "Anopheles" mosquito. It is responsible for roughly 50% of all malaria cases. It causes the disease's most dangerous form called falciparum malaria. It is therefore regarded as the deadliest parasite in humans, causing 435,000 deaths in 2017. It is also associated with the development of blood cancer (Burkitt's lymphoma) and is classified as Group 2A carcinogen.

The species originated from the malarial parasite "Laverania" found in gorillas, around 10,000 years ago. Alphonse Laveran was the first to identify the parasite in 1880, and named it "Oscillaria malariae". Ronald Ross discovered its transmission by mosquito in 1897. Giovanni Battista Grassi elucidated the complete transmission from a female anopheline mosquito to humans in 1898. In 1897, William H. Welch created the name "Plasmodium falciparum", which ICZN formally adopted in 1954. "P. falciparum" assumes several different forms during its life cycle. The human-infective stage are sporozoites from the salivary gland of a mosquito. The sporozoites grow and multiply in the liver to become merozoites. These merozoites invade the erythrocytes (RBCs) to form trophozoites, schizonts and gametocytes, during which the symptoms of malaria are produced. In the mosquito, the gametocytes undergo sexual reproduction to a zygote, which turns into ookinete. Ookinete forms oocyts from which sporozoites are formed.

As of the latest "World Malaria Report" of the World Health Organization, there were 219 million cases of malaria worldwide in 2017, up from 216 million cases in 2016. This resulted in an estimated 435,000 deaths. Almost every malarial death is caused by "P. falciparum", and 93% of death occurs in Africa. Children under five years of age are most affected, accounting for 61% of the total deaths. In Sub-Saharan Africa, over 75% of cases were due to "P. falciparum", whereas in most other malarial countries, other, less virulent plasmodial species predominate.

Falciparum malaria was familiar to the ancient Greeks, who gave the general name "pyretós" "fever". Hippocrates (c. 460-370 BCE) gave several descriptions on tertian fever and quartan fever. It was prevalent throughout the ancient Egyptian and Roman civilizations. It was the Romans who named the disease "malaria"—"mala" for bad, and "aria" for air, as they believed that the disease was spread by contaminated air, or miasma.

The British physician Patrick Manson formulated the mosquito-malaria theory in 1894; until that time, malarial parasites were believed to be spread in air as miasma, a Greek word for pollution. His colleague Ronald Ross, a British Army surgeon, travelled to India to test the theory. Ross discovered in 1897 that malarial parasite lived in certain mosquitoes. The next year, he demonstrated that a malarial parasite of birds could be transmitted by mosquitoes from one bird to another. Around the same time, Grassi demonstrated that "P. falciparum" was transmitted in humans only by female anopheline mosquito (in his case "Anopheles claviger"). Ross, Manson and Grassi were nominated for the Nobel Prize in Physiology or Medicine in 1902. Under controversial circumstances, only Ronald Ross was selected for the award.

There was a long debate on the taxonomy. It was only in 1954 the International Commission on Zoological Nomenclature officially approved the binominal "Plasmodium falciparum". The valid genus "Plasmodium" was created by two Italian physicians Ettore Marchiafava and Angelo Celli in 1885. The species name was introduced by an American physician William Henry Welch in 1897. It is derived from the Latin "falx", meaning "sickle" and "parum" meaning "like or equal to another".

"P. falciparum" is now generally accepted to have evolved from "Laverania" (a subgenus of "Plasmodium" found in apes) species present in gorilla in Western Africa. Genetic diversity indicates that the human protozoan emerged around 10,000 years ago. The closest relative of "P. falciparum" is "P. praefalciparum", a parasite of gorillas, as supported by mitochondrial, apicoplastic and nuclear DNA sequences. These two species are closely related to the chimpanzee parasite "P. reichenowi", which was previously thought to be the closest relative of "P. falciparum". "P. falciparum" was also once thought to originate from a parasite of birds.

Levels of genetic polymorphism are extremely low within the "P. falciparum" genome compared to that of closely related, ape infecting species of "Plasmodium" (including "P. praefalciparum")"." This suggests that the origin of "P. falciparum" in humans is recent, as a single "P. praefalciparum" strain became capable of infecting humans. The genetic information of "Plasmodium falciparum" has signaled a recent expansion that coincides with the agricultural revolution. It is likely that the development of extensive agriculture increased mosquito population densities by giving rise to more breeding sites, which may have triggered the evolution and expansion of "Plasmodium falciparum".

"P. falciparum" does not have a fixed structure but undergoes continuous change during the course of its life cycle. A sporozoite is spindle-shaped and 10-15 μm long. In the liver it grows into an ovoid schizont of 30-70 μm in diameter. Each schizont produces merozoites, each of which is roughly 1.5 μm in length and 1 μm in diameter. In the erythrocyte the merozoite form a ring-like structure, becoming a trophozoite. A trophozoites feed on the haemoglobin and forms a granular pigment called haemozoin. Unlike those of other "Plasmodium" species, the gametocytes of "P. falciparum" are elongated and crescent-shaped, by which they are sometimes identified. A mature gametocyte is 8-12 μm long and 3-6 μm wide. The ookinete is also elongated measuring about 18-24 μm. An oocyst is rounded and can grow up to 80 μm in diameter. Microscopic examination of a blood film reveals only early (ring-form) trophozoites and gametocytes that are in the peripheral blood. Mature trophozoites or schizonts in peripheral blood smears, as these are usually sequestered in the tissues. On occasion, faint, comma-shaped, red dots are seen on the erythrocyte surface. These dots are Maurer's cleft and are secretory organelles that produce proteins and enzymes essential for nutrient uptake and immune evasion processes.

The apical complex, which is actually a combination of organelles, is an important structure. It contains secretory organelles called rhoptries and micronemes, which are vital for mobility, adhesion, host cell invasion, and parasitophorous vacuole formation. As an apicomplexan, it harbours a plastid, an apicoplast, similar to plant chloroplasts, which they probably acquired by engulfing (or being invaded by) a eukaryotic alga and retaining the algal plastid as a distinctive organelle encased within four membranes. The apicoplast is involved in the synthesis of lipids and several other compounds and provides an attractive drug target. During the asexual blood stage of infection, an essential function of the apicoplast is to produce the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the MEP (non-mevalonate) pathway.

In 1995 the Malaria Genome Project was set up to sequence the genome of "P. falciparum". The genome of its mitochondrion was reported in 1995, that of the nonphotosynthetic plastid known as the apicoplast in 1996, and the sequence of the first nuclear chromosome (chromosome 2) in 1998. The sequence of chromosome 3 was reported in 1999 and the entire genome was reported on 3 October 2002. The roughly 24-megabase genome is extremely AT-rich (about 80%) and is organised into 14 chromosomes. Just over 5,300 genes were described. Many genes involved in antigenic variation are located in the subtelomeric regions of the chromosomes. These are divided into the "var", "rif", and "stevor" families. Within the genome, there exist 59 "var", 149 "rif", and 28 "stevor" genes, along with multiple pseudogenes and truncations. It is estimated that 551, or roughly 10%, of the predicted nuclear-encoded proteins are targeted to the apicoplast, while 4.7% of the proteome is targeted to the mitochondria.

Humans are the intermediate hosts in which asexual reproduction occurs, and female anopheline mosquitos are the definitive hosts harbouring the sexual reproduction stage.

Infection in humans begins with the bite of an infected female "Anopheles" mosquito. Out of about 460 species of "Anopheles" mosquito, more than 70 species transmit falciparum malaria. "Anopheles gambiae" is one of the best known and most prevalent vectors, particularly in Africa.

The infective stage called sporozoites released from the salivary glands through the proboscis of the mosquito enter the bloodstream during feeding. The mosquito saliva contains antihemostatic and anti-inflammatory enzymes that disrupt blood clotting and inhibit the pain reaction. Typically, each infected bite contains 20-200 sporozoites. The immune system clears the sporozoites from the circulation within 30 minutes. But a few escape and quickly invade liver cells (hepatocytes). The sporozoites move in the blood stream by gliding, which is driven by motor made up of proteins actin and myosin beneath their plasma membrane.

Entering the hepatocytes, the parasite loses its apical complex and surface coat, and transforms into a trophozoite. Within the parasitophorous vacuole of the hepatocyte, it undergoes 13-14 rounds of mitosis and meiosis which produce a syncytial cell (coenocyte) called a schizont. This process is called schizogony. A schizont contains tens of thousands of nuclei. From the surface of the schizont, tens of thousands of haploid (1n) daughter cells called merozoites emerge. The liver stage can produce up to 90,000 merozoites, which are eventually released into the bloodstream in parasite-filled vesicles called merosomes.

Merozoites use the apicomplexan invasion organelles (apical complex, pellicle and surface coat) to recognize and enter the host erythrocyte (red blood cell). The parasite first binds to the erythrocyte in a random orientation. It then reorients such that the apical complex is in proximity to the erythrocyte membrane. The parasite forms a parasitophorous vacuole, to allow for its development inside the erythrocyte. This infection cycle occurs in a highly synchronous fashion, with roughly all of the parasites throughout the blood in the same stage of development. This precise clocking mechanism has been shown to be dependent on the human host's own circadian rhythm.

Within the erythrocyte, the parasite metabolism depends on the digestion of hemoglobin. The clinical symptoms of malaria such as fever, anemia, and neurological disorder are produced during the blood stage.

The parasite can also alter the morphology of the erythrocyte, causing knobs on the erythrocyte membrane. Infected erythrocytes are often sequestered in various human tissues or organs, such as the heart, liver and brain. This is caused by parasite-derived cell surface proteins being present on the erythrocyte membrane, and it is these proteins that bind to receptors on human cells. Sequestration in the brain causes cerebral malaria, a very severe form of the disease, which increases the victim's likelihood of death.

After invading the erythrocyte, the parasite loses its specific invasion organelles (apical complex and surface coat) and de-differentiates into a round trophozoite located within a parasitophorous vacuole. The young trophozoite (or "ring" stage, because of its morphology on stained blood films) grows substantially before undergoing schizogony.

At the schizont stage, the parasite replicates its DNA multiple times and multiple mitotic divisions occur asynchronously. Each schizont forms 16-18 merozoites. The red blood cells are ruptured by the merozoites. The liberated merozoites invade fresh erythrocytes. A free merozoite is in the bloodstream for roughly 60 seconds before it enters another erythrocyte.

The duration of each blood stage is approximately 48 hours. This gives rise to the characteristic clinical manifestations of falciparum malaria, such as fever and chills, corresponding to the synchronous rupture of the infected erythrocytes.

Not all of the merozoites divide into schizonts; some get differentiated into sexual forms, male and female gametocytes. These gametocytes take roughly 7–15 days to reach full maturity, through the process called gametocytogenesis. These gametocytes are taken up by a female "Anopheles" mosquito during a blood meal.

The time of appearance of the symptoms from infection (called incubation period) is shortest for "P. falciparum" among "Plasmodium" species. An average incubation period is 11 days, but may range from 9 to 30 days. In isolated cases, prolonged incubation period as long as 2, 3 or even 8 years have been recorded. Pregnancy and co-infection with HIV are important conditions for delayed symptoms. Parasites can be detected from blood samples by the 10th day after infection (pre-patent period).

Within the mosquito midgut, the female gamete maturation process entails slight morphological changes, becoming more enlarged and spherical. The male gametocyte undergoes a rapid nuclear division within 15 minutes, producing eight flagellated microgametes by a process called exflagellation. The flagellated microgamete fertilizes the female macrogamete to produce a diploid cell called a zygote. The zygote then develops into an ookinete. The ookinete is a motile cell, capable of invading other organs of the mosquito. It traverses the peritrophic membrane of the mosquito midgut and crosses the midgut epithelium. Once through the epithelium, the ookinete enters the basal lamina, and settles to an immotile oocyst. For several days, the oocyst undergoes 10 to 11 rounds of cell division to create a syncytial cell (sporoblast) containing thousands of nuclei. Meiosis takes place inside the sporoblast to produce over 3,000 haploid daughter cells called sporozoites on the surface of the mother cell. Immature sporozoites break through the oocyst wall into the haemolymph. They migrate to the mosquito salivary glands where they undergo further development and become infective to humans.

A single anopheline mosquito can transmit hundreds of "P. falciparum" sporozoites in a single bite under experimental conditions. But in nature the number is generally less than 80. The sporozoites do not enter the blood stream directly and remain in the skin tissue for 2 to 3 hours. About 15–20% sporozoites enter the lymphatic system where they activate dendritic cells, which send them for destruction by T lymphocytes (CD8+ T cells). At 48 hours after infection, "Plasmodium"-specific CD8+ T cells can be detected in the lymph nodes connected to the skin cells. Most of the sporozites remaining in the skin tissue are subsequently killed by the innate immune system. The sporozoite glycoprotein specifically activates mast cells. The mast cells then produce signalling molecules such as TNFα and MIP-2, which activate cell eaters (professional phagocytes) such as neutrophils and macrophages.

Only a small number (0.5-5%) of sporozoites enter the blood stream into the liver. In the liver, the activated CD8+ T cells from the lymph bind the sporozoites through the circumsporozoite protein (CSP). Antigen presentation by dendritic cells in the skin tissue to T cells is also a crucial process. From this stage onward the parasites produce different proteins that help in suppressing communication of the immune cells. Even at the height of the infection when RBCs are ruptured, the immune signals are not strong enough to activate macrophages or natural killer cells.

Although "P. falciparum" is easily recognized by human immune system while in the bloodstream, it evades immunity by producing over 2,000 cell membrane antigens The initial infective stage sporozoites produce circumsporozoite protein (CSP), which binds to hepatocytes. Binding to and entry into the hepatocytes is aided by another protein, thrombospondin-related anonymous protein (TRAP). TRAP and other secretory proteins (including sporozoite microneme protein essential for cell traversal 1, SPECT1 and SPECT2) from microneme allow the sporozoite to move through the blood, avoiding immune cells and penetrating hepatocytes.

During erythrocyte invasion, merozoites release merozoite cap protein-1 (MCP1), apical membrane antigen 1 (AMA1), erythrocyte-binding antigens (EBA), myosin A tail domain interacting protein (MTIP), and merozoite surface proteins (MSPs). Of these MSPs, MSP1 and MSP2 are primarily responsible for avoiding immune cells. The virulence of "P. falciparum" is mediated by erythrocyte membrane proteins, which are produced by the schizonts and trophozoites inside the erythrocytes and are displayed on the erythrocyte membrane. PfEMP1 is the most important, capable of acting as both an antigen and an adhesion molecule.

The clinical symptoms of falciparum malaria are produced by the rupture of schizont and destruction of erythrocytes. Most of the patients experience fever (>92% of cases), chills (79%), headaches (70%), and sweating (64%). Dizziness, malaise, muscle pain, abdominal pain, nausea, vomiting, mild diarrhea, and dry cough are also generally associated. High heartrate, jaundice, pallor, orthostatic hypotension, enlarged liver, and enlarged spleen are also diagnosed.

"P. falciparum" works via sequestration, a distinctive property not shared by few other "Plasmodiae". The mature schizonts change the surface properties of infected erythrocytes, causing them to stick to blood vessel walls (cytoadherence). This leads to obstruction of the microcirculation and results in dysfunction of multiple organs, such as the brain in cerebral malaria.

"P. falciparum" is responsible for (almost) all severe human illnesses and deaths due to malaria, in a condition called complicated or severe malaria. Complicated malaria occurs more commonly in children under age 5, and sometimes in pregnant women (a condition specifically called pregnancy-associated malaria). Women become susceptible to severe malaria during their first pregnancy. Susceptibility to severe malaria is reduced in subsequent pregnancies due to increased antibody levels against variant surface antigens that appear on infected erythrocytes. But increased immunity in mother increases susceptibility to malaria in newborn babies.

"P. falciparum" is found in all continents except Europe. According to the WHO "World Malaria Report 2018", 219 million people suffered from malaria in 2017, an increase from 216 million in 2016. 435,000 people died from it. The infection is most prevalent in Africa, where 92% of malaria deaths occr. Children under five years of age are most affected and 61% of malaria deaths occurred in this age group. 80% of the infection is found in Sub-Saharan Africa, 7% in the South-East Asia, and 2% in the Eastern Mediterranean. Nigeria has the highest incidence with 27% of the total global cases. Outside Africa, India has the highest incidence with 4.5% of the global burden. Europe is regarded as a malaria-free region. Historically, the parasite and its disease had been most well known in Europe. But medical programmes, such as insecticide spraying, drug therapy and environmental engineering since the early 20th century resulted in complete eradication in the 1970s. It is estimated that approximately 2.4 billion people are at constant risk of infection.

In 1640, Huan del Vego first employed the tincture of the cinchona bark for treating malaria; the native Indians of Peru and Ecuador had been using it even earlier for treating fevers. Thompson (1650) introduced this "Jesuits' bark" to England. Its first recorded use there was by John Metford of Northampton in 1656. Morton (1696) presented the first detailed description of the clinical picture of malaria and of its treatment with cinchona. Gize (1816) studied the extraction of crystalline quinine from the cinchona bark and Pelletier and Caventou (1820) in France extracted pure quinine alkaloids, which they named quinine and cinchonine. The total synthesis of quinine was achieved by American chemists R.B. Woodward and W.E. Doering in 1944. Woodward received the Nobel Prize in Chemistry in 1965.

Attempts to make synthetic antimalarials began in 1891. Atabrine, developed in 1933, was used widely throughout the Pacific in World War II, but was unpopular because of its adverse effects. In the late 1930s, the Germans developed chloroquine, which went into use in the North African campaigns. Creating a secret military project called Project 523, Mao Zedong encouraged Chinese scientists to find new antimalarials after seeing the casualties in the Vietnam War. Tu Youyou discovered artemisinin in the 1970s from sweet wormwood ("Artemisia annua"). This drug became known to Western scientists in the late 1980s and early 1990s and is now a standard treatment. Tu won the Nobel Prize in Physiology or Medicine in 2015.

According to WHO guidelines 2010, artemisinin-based combination therapies (ACTs) are the recommended first-line antimalarial treatments for uncomplicated malaria caused by "P. falciparum". WHO recommends combinations such as artemether/lumefantrine, artesunate/amodiaquine, artesunate/mefloquine, artesunate/sulfadoxine-pyrimethamine, and dihydroartemisinin/piperaquine.

The choice of ACT is based on the level of resistance to the constituents in the combination. Artemisinin and its derivatives are not appropriate for monotherapy. As second-line antimalarial treatment, when initial treatment does not work, an alternative ACT known to be effective in the region is recommended, such as artesunate plus tetracycline or doxycycline or clindamycin, and quinine plus tetracycline or doxycycline or clindamycin. Any of these combinations is to be given for 7 days. For pregnant women, the recommended first-line treatment during the first trimester is quinine plus clindamycin for 7 days. Artesunate plus clindamycin for 7 days is indicated if this treatment fails. For travellers returning to nonendemic countries, atovaquone/proguanil, artemether/lumefantrineany and quinine plus doxycycline or clindamycin are recommended.

For adults, intravenous (IV) or intramuscular (IM) artesunate is recommended. Quinine is an acceptable alternative if parenteral artesunate is not available.

For children, especially in the malaria-endemic areas of Africa, artesunate IV or IM, quinine (IV infusion or divided IM injection), and artemether IM are recommended.

Parenteral antimalarials should be administered for a minimum of 24 hours, irrespective of the patient's ability to tolerate oral medication earlier. Thereafter, complete treatment is recommended including complete course of ACT or quinine plus clindamycin or doxycycline.

RTS,S is the only candidate as malaria vaccine to have gone through clinical trials. Analysis of the results of the phase III trial (conducted between 2011 and 2016) revealed a rather low efficacy (20-39% depending on age, with up to 50% in 5–17-month aged babies), indicating that the vaccine will not lead to full protection and eradication.

The International Agency for Research on Cancer (IARC) has classified malaria due to "P. falciparum" as Group 2A carcinogen, meaning that the parasite is probably a cancer-causing agent in humans. Its association with a blood cell (lymphocyte) cancer called Burkitt's lymphoma is established. Burkit's lymphoma was discovered by Denis Burkitt in 1958 from African children, and he later speculated that the cancer was likely due to certain infectious diseases. In 1964, a virus, later called Eppstein-Barr virus (EBV) after the discoverers, was identified from the cancer cells. The virus was subsequently proved to be the direct cancer agent, and is now classified as Group 1 carcinogen. In 1989, it was realised that EBV requires other infections such as with malaria to cause lymphocyte transformation. It was reported that the incidence of Burkitt's lymphoma decreased with effective treatment of malaria over several years. The actual role played by "P. falciparum" remained unclear for the next two-and-half decades. EBV had been known to induce lymphocytes to become cancerous using its viral proteins (antigens such as EBNA-1, EBNA-2, LMP-1, and LMP2A). From 2014, it became clear that "P. falciparum" contributes to the development of the lymphoma. "P. falciparum"-infected erythrocytes directly bind to B lymphocytes through the CIDR1α domain of PfEMP1. This binding activates toll-like receptors (TLR7 and TLR10) causing continuous activation of lymphocytes to undergo proliferation and differentiation into plasma cells, thereby increasing the secretion of IgM and cytokines. This in turn activates an enzyme called activation-induced cytidine deaminase (AID), which tends to cause mutation in the DNA (by double-strand break) of an EBV-infected lymphocytes. The damaged DNA undergoes uncontrolled replication, thus making the cell cancerous.

The high mortality and morbidity caused by "P. falciparum" has placed great selective pressure on the human genome. Several genetic factors provide some resistance to "Plasmodium" infection, including sickle cell trait, thalassaemia traits, glucose-6-phosphate dehydrogenase deficiency, and the absence of Duffy antigens on red blood cells. The presence of "P. falciparum" in human populations has exerted selective pressure on the human genome. E. A. Beet, a doctor working in Southern Rhodesia (now Zimbabwe) had observed in 1948 that sickle-cell disease was related to lower rate of malaria infection. This suggestion was reiterated by J. B. S. Haldane in 1948, who suggested that thalassaemia could provide similar protection. This hypothesis has since been confirmed and extended to hemoglobin E, hemoglobin C and Hemoglobin S.

Klebs made some significant errors about infectious diseases. He believed, for example, that malaria was caused by a bacterium. In 1879, Corrado Tommasi-Crudeli and he claimed that they isolated a bacterium from the waters of Pontine Marshes in Roman Campagna. They concluded that the bacterium was the pathogen for malaria as they discovered it from damp soil in the region of malaria epidemics. They gave it the name "Bacillus malariae". They further experimented with the bacterial isolate which they injected into rabbits. They observed that infected rabbits developed fever and enlarged spleen, characteristics of malaria. They proposed that the malarial bacterium was transmitted by drinking contaminated water or inhalation from air. Klebs reported that antimalarial drug quinine killed the germ. The discovery was supported by leading malariologists of the time. It was then declared that the malaria problem was solved. When a French Army physician Charles Alphonse Laveran correctly discovered in 1880 that malaria was caused by a protozoan parasite (which he called "Oscillaria malariae", now "Plasmodium falciparum"), the discovery was ignored in preference of the bacillus theory of Klebs and Tommasi-Crudeli. An American physician, though, George Miller Sternberg, proved that the bacillus did not cause specific symptoms of malaria in 1881. The bacillus theory was eventually proved wrong by the experimental demonstration of the mosquito-malaria theory in 1898.

Edwin Klebs

Theodor Albrecht Edwin Klebs (6 February 1834 – 23 October 1913) was a German-Swiss pathologist. He is mainly known for his work on infectious diseases. His works paved the way for the beginning of modern bacteriology, and inspired Louis Pasteur and Robert Koch. He was the first to identify a bacterium that causes diphtheria, which was called Klebs–Loeffler bacterium (now "Corynebacterium diphtheriae"). He was the father of physician Arnold Klebs.

Klebs was born in Königsberg, Province of Prussia. He studied at the University of Würzburg under Rudolf Virchow in 1855 and received his doctorate at the University of Berlin in 1858. He achieved his habilitation at the University of Königsberg the following year.

Klebs was an assistant to Virchow at the Charité in Berlin from 1861 until 1866, when he became a professor of pathology at the University of Bern in Switzerland. He married Rosa Grossenbacher, a Swiss, and also acquired Swiss citizenship. He served as a military physician for the Prussian Army in 1870 during the Franco-Prussian War; several of his ancestors had fought during the Napoleonic Wars.

Klebs taught at Würzburg from 1872 to 1873, at Prague from 1873 to 1882, and at Zürich from 1882 to 1892. Because of disagreements with the rest of the faculty, the impetuous Klebs resigned from Zürich in 1893 and ran an unsuccessful private business in Karlsruhe and Strassburg in 1894.

From 1896 to 1900, Klebs taught at Rush Medical College in Chicago, United States. From 1905 to 1910, he was a private researcher in Berlin, after which he returned to Switzerland, living with his oldest son in Lausanne. Klebs died in Bern.

In 1883, Klebs successfully identified the bacterium "Corynebacterium diphtheriae" as the etiological cause of diphtheria. This bacterium is also known as the Klebs-Löffler bacillus.

The bacterial genus "Klebsiella" is named in honor of his work.

Klebs' works preceded some of the most important discoveries in medicine. He described acromegaly in 1884, two years before Pierre Marie. In 1878, he successfully inoculated syphilis in monkeys, antedating Élie Metchnikoff and Émile Roux by 25 years. He isolated colonies of bacteria nine years before Robert Koch. He was the first to produce tuberculosis experimentally in animals by the injection of milk from infected cows. He identified the typhoid bacillus (now named "Salmonella typhi") before Karl Joseph Eberth.

Klebs identified four ""Grundversuche"" (fundamental tests) that provided a basis for his own research strategy, as well as general bacteriological research. According to Klebs, the bacteriological tests consist of the following postulates:

Although some of these hypotheses are literally false, they are in general the foundation of modern experiments in bacteriology.

Klebs also made mistakes in claiming the existence of "Microzoon septicum" as causative agent of wound infection, and "monadines" as the pathogen for rheumatism.

Mosquito-malaria theory (or sometimes mosquito theory) was a scientific theory developed in the latter half of the 19th century that solved the question of how malaria was transmitted. The theory basically proposed that malaria was transmitted by mosquitoes, in opposition to the centuries-old medical dogma that malaria was due to bad air, or miasma. The first scientific idea was postulated in 1851 by Charles E. Johnson, who argued that miasma had no direct relationship with malaria. Although Johnson's hypothesis was forgotten, the arrival and validation of the germ theory of diseases in the late 19th century began to shed new lights. When Charles Louis Alphonse Laveran discovered that malaria was caused by a protozoan parasite in 1880, the miasma theory began to subside.

Mosquito-malaria theory

An important discovery was made by Patrick Manson in 1877 that mosquito could transmit human filarial parasite. Inferring from such novel discovery Albert Freeman Africanus King proposed the hypothesis that mosquitoes were the source of malaria. In the early 1890s Manson himself began to formulate the complete hypothesis, which he eventually called the mosquito-malaria theory. According to Manson malaria was transmitted from human to human by a mosquito. The theory was scientifically proved by Manson's confidant Ronald Ross in the late 1890s. Ross discovered that malaria was transmitted by the biting of specific species of mosquito. For this Ross won the Nobel Prize for Physiology or Medicine in 1902. Further experimental proof was provided by Manson who induced malaria in healthy human subjects from malaria-carrying mosquitoes. Thus the theory became the foundation of malariology and the strategy of control of malaria.

Malaria was prevalent in the Roman Empire, and the Roman scholars associated the disease with the marshy or swampy lands where the disease was particularly rampant. It was from those Romans the name "malaria" originated. They called it malaria (literally meaning "bad air") as they believed that the disease was a kind of miasma that was spread in the air, as originally conceived by Ancient Greeks. Then it was a medical consensus that malaria was spread due to miasma, the bad air. The first record of argument against the miasmatic nature of malaria was from an American physician John Crawford. Gorgas wrote an article "Mosquital Origin of Malarial Disease" in "Baltimore Observer" in 1807, but it provoked no consequences. An American physician Charles Earl Johnson provided a systematic and elaborate arguments against miasmatic origin of malaria in 1851 before the Medical Society of North Carolina. Some of his important points were:

The notion that malaria was due to miasma was negated by the discovery of malarial parasite. A German physician Johann Heinrich Meckel was the first to observed in 1847 the protozoan parasites as black pigment granules from the blood and spleen of a patient who died of malaria. But he did not understand the parasitic nature and significance of those granules in connection with malaria. In 1849 a German pathologist Rudolf Virchow realised that it could be those granules that were responsible for the disease. In 1879 an Italian biologist Ettore Afanasiev further argued that the granules were definitely the causative agents.
A major discovery was made by a French Army physician Charles Louis Alphonse Laveran working in Algeria, North Africa. At the hospital in Bône (now Annaba), he noticed spherical bodies from a patient’s blood film, free or adherent to red blood cells. On 6 November 1880 he observed from one patient's blood the actual living parasite, describing it as "a pigmented spherical body, filiform elements which move with great vivacity, displacing the neighboring red blood cells." He also observed the process of maturation of the parasite (which is now called exflagellation of microgametocytes). He meticulously examined 200 patients, and noted the cellular bodies in all 148 cases of malaria but never in those without malaria. He also found that after treatment with quinine, the parasites disappeared from blood. These findings clearly indicated that the parasite was the cause of malaria, and establishing the germ theory (nature) of malaria. He named the parasite "Oscillaria malariae" (later renamed "Plasmodium malariae") and reported his discovery to the French Academy of Medicine in Paris on 23 November and 28 December. For his discovery he was awarded the Nobel Prize for Physiology or Medicine in 1907.

In the early 1880s Laveran's germ theory of malaria was generally accepted by the science community. However pivotal problems still remained, such as what transmit the malarial parasites and how. The scientific clue emerged when a British medical officer Patrick Manson discovered for the first time that parasites were transmitted by mosquitoes. In 1877 while working in Amoy, a coastal town in China, he found that the mosquito "Culex fatigans" (now "Culex quinquefasciatus") was the vector of the filarial roundworm that he called "Filaria sanguinis hominis" (but now "Wuchereria bancrofti"). His findings were published in the "China Customs Medical Report" in 1878, and relayed by Spencer Cobbold to the Linnean Society in London. This was the first direct evidence that mosquitoes could transmit microscopic parasites in humans, further suggesting that the same could be true in case of malaria.

Based on the report of Manson's discovery, an American physician Albert Freeman Africanus King developed a proposition that malaria is transmitted by mosquitoes. He revealed his idea in 1881 to his colleagues C.V. Riley and L.O. Howard, who did not share the same opinion. Unfettered he developed the theory with proper justifications and presented it before the Philosophical Society of Washington on 10 February 1882, under the title "The Prevention of Malarial Disease Illustrating inter alia the Conservative Function of Ague". He went so far as to suggest the complete covering of Washington, DC along the Washington Monument with giant net to protect the city from malaria. His idea was ridiculed as inconceivable as scientist still believed malarial parasite was spread through inhalation or ingestion from air (still not far from the miasma theory). He did not give up, and instead formed a more elaborate argument which he published as a 15-page article in the September 1883 issue of "The Popular Science Monthly", making an introduction as:
King carefully selected his view in 19 points. To paraphrase his lengthy arguments: occurrence of malaria always coincided with conditions that are also ideal for mosquitos, such as in the time of day, geographical area, temperature, and climate. But the flaw in his proposition was that he believed malaria was transmitted by mosquito through its eggs.

In 1889 Patrick Manson returned to England and worked at the Seamen's Hospital Society and also as lecturer on tropical diseases in St George's Hospital at London in 1882. His attention was soon drawn towards malaria and began to realise the implications of his own discovery of filarial transmission on malaria. He strongly supported Laveran's germ theory of malaria, which was not yet completely embraced by the entire medical community of the time. He proposed that:
Manson was unfortunate that he could not investigate his theory as he was not in malaria endemic country such as India, where it could be experimentally proven. But fortunately he met a British army surgeon Ronald Ross, who was on vacation while serving in the Indian Medical Service in India. In November 1894, he revealed to Ross with his hands on Ross' shoulders, saying, "Do you know, I have formed the theory that mosquitoes carry malaria just as they carry filaria."

In 1894 Patrick Manson devised an ingenious procedure for detecting malarial parasites at different developmental stages from blood samples. This would later proved to be the tool for experimental proof of his theory. Manson demonstrated to and taught Ronald Ross the technique from which Ross became convinced of Laveran's germ theory. Trained and mentored by Manson, Ross returned to India in March 1895 to start his investigation. But to the dismay of Ross it was not an easy task. His first detection of malarial parasite from patients came only after two months of hard work. The disappointed Ross had to be encouraged by Manson calling the study as the "Holy Grail" of malaria research, and that Ross was the "Sir Galahad". After one and half years he made no significant progress. On 20 August 1897 he made a momentous discovery that some mosquitoes had malarial parasites in them. He had fed the blood of a malarial patient (Husein Khan) to different groups of mosquitoes four days before, and found that only one type (which he called "brown type" or more commonly "dappled-winged mosquitoes", not knowing the species, which in fact was "Anopheles") acquired the malarial parasites in its stomach. This was the first evidence for Manson's theory that mosquito did carry the malarial parasite, and Ross would later famously call 20 August as "Malaria Day" (now adopted as World Mosquito Day).

The second experimental evidence came in the mid-1898 when Ross demonstrated the transmission of bird malaria "Proteosoma relictum" (now "Plasmodium relictum") between larks and mosquitoes, which he called "grey mosquitos" (which were "Culex fatigans", but now renamed "Culex quinquefasciatus"). He showed that the mosquitoes ingested the parasites from infected birds and could infect healthy birds. He further discovered that the parasites developed in the stomach wall and were later stored in salivary glands of the mosquito. This was a conclusive evidence that malarial parasites were indeed transmitted by mosquitoes. In his report Ross concluded that:

On 9 July 1898 Ross wrote Manson:
Ross' scientific evidences were soon fortified by Italian biologists including Giovanni Battista Grassi, Amico Bignami, and Giuseppe Bastianelli, who discovered that human malarial parasite was transmitted by the actual biting (disproving one of Manson's hypotheses) of female mosquito. In 1899 they reported the infection of "Plasmodium falciparum" with the mosquito "Anopheles claviger" However the practical importance of validating the theory, i.e. control of mosquito vector should be an effective management strategy for malaria, was not realised by the medical community and the public. Hence in 1900 Patrick Manson clinically demonstrated that the bite of infected anopheline mosquitoes invariably resulted in malaria. He acquired carefully reared infected mosquitoes from Bignami and Bastianelli in Rome. His volunteer at the London School of Tropical Medicine, P. Thurburn Manson gave a detailed account of his malarial fevers and treatment after bitten by the mosquitoes. As he summarised, Manson's clinical trial showed that the practical solution to malaria infection was in: