In Pakistan, malaria is still a major public health issue despite significant progress. Malaria incidence exhibits considerable variation across different regions, making accurate analysis challenging due to insufficient information [37]. The health departments of four provinces have consistently reported rising annual malaria incidence rates since the late 1970s. The provinces of Sindh and KP reported increasing levels of malaria incidence across the entire province [16, 38].
Pakistan experiences extreme weather conditions with all four seasons due to which malaria parasites have uneven distribution all over country and their prevalence varies with the seasons. [16, 39]. As a result, determining the precise prevalence of malaria infection in Pakistan has been proved difficult [16]. It is noticed that KP is the most disastrous hit province of Pakistan compared to the other provinces [16]. This study was commenced to precisely estimate the occurrence of malaria from 10 districts of KP, Pakistan.
A total of 13363 suspected cases of malaria were found throughout the current study in which 1004 were found microscopically positive. Among all positive cases, the Southern zone (n = 539) has the highest malaria-positive cases than the Northern zone (n = 465).
The collective slide positivity rate (SPR) across all enlisted disricts of Khyber Pakhtunkhwa (KP) was 7.52% however, the SPR was higher in Southern KP (8.16%) in contrast to the Northern KP (6.88%). Qureshi et al. [10] documented SPR of 5.7% in two distinct areas of Punjab. Yasinzai and Kakarsulemankhel [26] examined the slide positivity rate, ranging from 19.8 to 58.1% across different age categories. Mahmood, [40] reported 35% SPR in 348 patients with malaria positive in Karachi. Leghari et al. [41] observed SPR in malaria patients from 0.33 to 0.22% in Bahawalpur City only from 2007 to 2011. Similarly, the annual parasite incidence (API) was also high in Southern KP in contrast to Northern KP (0.1 > 0.04) per 1000 population. Another study by Qureshi et al. [10] reported API from two distinct zones of Punjab and found high in Southern Punjab (0.2 per 1000 population) in contrast to Northern Punjab (0.1 per 1000 population). Additionally, Leghari et al. [41] conducted a five year malaria surveillance in Bahawalpur city from 2007 to 2011, revealing an API ranging from 0.07 to 0.1 per 1000 population. The ABER in the current study was also high in Southern KP (0.14%) in contrast to the Northern KP (0.05%). Similarly Qureshi et al. [10] also observed ABER high in Southern Punjab (0.26%) in contrast to Northern Punjab (0.17%).
Understanding the seasonal pattern of malaria parasitemia is essential for the strategic planning and effective implementation of integrated interventions for malaria control. This study revealed fluctuation in the prevalence of malaria parasitemia throughout the seasons in Khyber Pakhtunkhwa. Particularly, the highest prevalence and density of P. vivax were observed during the monsoon and post-monsoon periods. This finding unveils novel seasonal patterns, indicating duration somewhat longer than those identified in a prior study conducted by Prybylski [42] under a USAID initiative, in the province of Punjab. They observed that the prevalence of P. vivax was more pronounced in the initial months from July to August. The elevated temperature difference in KP from the preceding decade, attributed to global warming, and post-monsoon are believed to contribute to the seasonal variation in the dominance of P. vivax. Similarly, distribution patterns are known to exist in certain areas of Punjab Pakistan, and neighboring country Afghanistan that share similar seasonal and climate changes [10, 43].
The increase in malaria cases during the spring months (March to May) can be attributed to the extended summer period and enhanced mosquito breeding conditions resulting from these seasonal variations. However, malaria cases are at their lowest from December to February (the winter season) due to changes in climatic conditions such as a change in rainfall and temperature and unstable vector dynamics [8]. In this study, the age-wise distribution of malaria was estimated as well. All recruited cases, of both sexes ranged in age from 1 to 100 years. The age range from 1 to 15 years had a higher incidence than the age range of 16 to 30 years and 31 to 45 years, respectively. A similar finding is observed in a study conducted in Niger where malaria was maximum among the age groups 2–10 years (48%) [44]. Similarly Karim et al. [45] investigated the occurrence of clinical malaria in the tribal districts of Pakistan spanning from March 2016 to December 2018. Their finding reviled that individuals infected with both Plasmodium species predominantly belong to the age group of 1 to 15 years old. This implies that the heightened susceptibility of the age range from 1 to 15 years can be attributed to factors, such as low acquired immunity, increased engagement in outdoor activities, and greater exposure to vectors. Another significant contributing factor to the incidence with in this age group is the large proportion (65%) of Pakistan total population falling within this demographic. Conversely malaria cases were observed to be less prevalent in the older age groups (46->60), potentially associated with their lower exposure to infected mosquito bites due to good awareness about malaria and control prevention practices.
In the current investigation malaria effect a greater number of males, with 740 (73.70%) cases, compared to females, accounting for 264 (26.29%) cases across both zones of Khyber Pakhtunkhwa. This finding correlates with studies in two different zones of Punjab, where a higher prevalence of malaria was documented among males (71%) compared to females (31%). [10]. Similarly Khattak et al. [16] also reported that males (64%) were more infected than females (36%). Our study is in line with a previous study in tribal districts of Pakistan conducted from 2016 to 2018 where they found high cases of malaria in males (74%) as compared to females (26%) [45]. The fact that the male population typically participates in outdoor activities during dusk and dawn, when exophagic mosquito species are most active, may account for the greater incidence rate in males. Moreover, males tend to engage in seasonal migration for various activities such as trade, agriculture, and other occupational risks particularly to regions with higher malaria risks. This behavior exposes them to infective mosquito bites and increases their susceptibility to contracting malaria. In Pakistan, various findings from various regions indicated a higher likelihood of malaria contraction among males compared to females [46, 47] which are similar to the outcomes observed in the current study. The variation in malaria transmission rates based on gender is likely a result of Pakistan’s socioeconomic conditions and the limited involvement of females in agriculture activities.
Among the 1004 positive cases, P. vivax accounted for 78.3% of malaria infections, P. falciparum for 17.2%, and mixed infections for 4.3% as determined by microscopy. However, through molecular analysis, 62.8% were P. vivax, 10.9% were P. falciparum and 9.9% were mixed species. However, out of the 163 samples positive under microscopy (16.23%), they did not successfully amplify in the molecular analysis. This could be attributed to potential DNA damage caused by the anticoagulant used in EDTA tubes or false-positive results from microscopy. While microscopy results indicated 44 samples as mixed infections, PCR analysis identified 100 samples with mixed infections. The PCR result revealed that several species initially identified microscopically either P. vivax or P. falciparum were, in fact, mixed infections. The current findings also showed that Northern KP had a lower rate of mixed infection than Southern KP. Qureshi et al. [10] reviled the incidence of malaria in two different zones (Southern and Northern zones) of Punjab and reported P. vivax (66.7% ), P. falciparum (23.7%), and mixed infections (9.6%) through microscopy out of a total of 925 positive cases and through PCR 53.40% were P. vivax, 18.7%, P. falciparum, and 12.7% were mixed species. our finding is congruent with another similar study in tribal districts of Pakistan and reported P. vivax (68%) and P. falciparum (11% ) through microscopy while P. vivax (87%) and P. falciparum (13% ) through nPCR analysis [45]. Similarly Sheikh et al. [48] studied the malaria endemicity in Quetta City of Pakistan and found 35% positive cases with 30.7% P. falciparum and 66.8% P. vivax infections. Khattak et al. [16] investigated Plasmodium parasitemia investigation from four provinces of Pakistan and reported 76% P. vivax, 18% P. falciparum, and 6% mixed infections in 2011. However, they covered only three cities in Khyber Pakhtunkhwa and reported 72% P. vivax, 20% P. falciparum, and 8% mixed infections. One malaria species' dominance over another is mostly influenced by the parasitology biology as well as local climatic and seasonal changes because P. falciparum does not experience real relapses while relapses are common in P. vivax due to the Vivax hypnozoite's longer survival in liver cells [8]. In the present findings, out of ten major districts of KP highest malaria incidence was found in D.I. Khan i.e., (6.97%) followed by Hangu (6.92%) from the Southern zone and Charsadda (6.33%) followed by Mardan (6.08%) from Northern zone. Likewise Qureshi et al. [10] studied a high incidence of malaria in Rajanpur (21.4%) from the Southern zone and Rawalpindi (25.5%) from the Northern zone in 10 different cities of Punjab.
Ahmad et al. [49] observed that malaria infections in Lal Qilla Lower Dir, KP, were predominantly attributed to P. vivax at 99.4% with only 0.53% attributed to P. falciparum. our finding is congruent with another similar study in tribal districts of Pakistan which reported 68% P. vivax and 11% P. falciparum through microscopy while 87% P. vivax and 13% P. falciparum through nPCR analysis [45]. In contrast, a report from Baqai Medical University Hospital in Karachi, Pakistan, in 2002 indicated a higher prevalence of P. falciparum (65%) in comparison to P. vivax (35%) among malaria positive children [50]. Similarly Mehmood et al. [40] similarly examined a high percentage of P. falciparum cases (88.5%) in Karachi, Pakistan, in comparison to P. vivax cases (9%). None of the samples from the selected districts showed the presences of P. malariae or P. ovale. Mixed species infections are majorly prevalent in the middle and southwest coast of India and all four provinces of Pakistan. Additionally, other tropical malaria-endemic countries like Cambodia, Thailand, India, and many others, have frequently reported cases of mixed malaria infection [51]. Since P. vivax malarial-infection is higher than P. falciparum in the present study. It may be due to an increase in humidity and temperature conditions from Northern to Southern Khyber Pakhtunkhwa (KP), Pakistan.
According to socioeconomic standards, WHO, [7] observed that Pakistan's rural areas had a higher malaria infection rate. The current findings also showed that, in comparison to Northern KP districts, malaria was more common in Southern KP districts due to a lack of health care services, low development, and the use of presumed treatment. The ongoing monsoon season in 2022 has inflicted substantial damage in Pakistan, with record rainfall reaching three times the usual annual average [52]. Environmental conditions, notably the extensive flooding following exceptional monsoon rains in Southern KP, have expanded the suitable zones for mosquito vector proliferation and breeding, consequently amplifying malaria incidences. It is a well-known fact that the prevalence of malaria rises after flooding. The onset of floods at the start decreases mosquito breeding. However, once the floods recede, the massive amount of stagnant water left behind functioning as a mosquito nursery, allows the mosquitoes that transmit malaria to grow and breed in huge numbers.
In all districts of Northern KP, P. vivax was highly prevalent, with Peshawar district having the highest occurrence of mixed species. Similarly, Southern KP exhibited a high prevalence of P. vivax, and in Bannu district, P. falciparum emerged as the predominant species. The largest human migration has resulted from army action against terrorists in KP. About 3.4 million IDPs (Internally displaced persons) were displaced [39, 53]. The medical problems and other needs of life for these IDPs living in camps were completely different from natural calamities. IDPs camps are densely crowded with people, have terrible hygienic conditions, and serve contaminated water. The likelihood of human-vector contact increases due to overcrowding. Pits excavated for latrines or to remove soil for building shelters trap standing water, which gives the anopheles vector additional opportunities to breed. The contact between mosquito vectors and humans is also increases because a refugee camp's surroundings are typically devoid of vegetation, providing limited places for mosquitoes to rest [54]. IDPs are more susceptible to illnesses. Malaria is the primary physical health issue for IDPs. Recently IDPs have left high-malaria endemic areas like North Waziristan, the Pak-Afghan border, and FATA [4, 55] of Southern KP due to anti-terrorism military operation, has led to an increased incidence of malaria infections in Southern KP.
The evolutionary relationship of the isolates of P. falciparum and P. vivax appears to be widespread in nature and is not restricted to any particular region. The isolates of P. vivax from Southern and Northern KP show more diversity from Southern parts and show homology with the P. vivax isolates of China while India and Iran have slightly less resemblance with our current study. The P. falciparum isolates in our study exhibited similarity with those from India and China, indicating homology. However, our findings suggest that P. falciparum isolates from Mexica and Kuwait, despite sharing common ancestors, displayed a slight lower resemblance. A similar type origin of P. falciparum was studied and observed by Qureshi et al. [10] and Joy et al. [56]. In contrast, P. vivax exhibited a population with greater diversity compared to P. falciparum. A large increase in humidity and temperature causes an increase in the rate at which vectors breed and their vectorial capability, which may promote the spread of Plasmodium and its resurgence [55, 57]. The anopheles mosquito population in the area has adapted to various breeding environments, with Anopheles culicifacies favoring sewage drains, An. fluviatilis breeding in streams, and An. Stephensi primarily reproducing in clear water [58–60], as P. vivax is majorly prevalent in hilly, rural, and cultivated regions, similarly, all ten selected districts have cultivated areas of rice and sugarcane, making these recruited districts suitable to parasite breeding [10, 55].
Our investigation revealed a considerable discrepancy between microscopy and PCR diagnosis. Microscopic examination, a conventional and cost-effective method for detecting Plasmodium species, is characterized by being less time consuming. Although other factors like the microscope's condition, the staining pattern on the slide, the number of parasites in the blood, and the experience of the technicians also have an impact on the accuracy of the results. PCR is the most effective method for diagnosing any of the five Plasmodium species but PCR is more expensive than microscopy. The Orakzai agency's epidemiological investigation of malaria revealed that microscopy had a greater positive result than PCR [55, 61]. Shahzadi et al. [62] investigated optimal malaria diagnostic methods and found that PCR-based diagnostic exhibit higher sensitivity, specificity, and accuracy compared to microscopy, which serves as the standard for routine laboratory malaria diagnosis. Microscopy is not a sensitive diagnostic method when dealing with mixed infections and low parasite densities. Similarly, Qureshi et al. [10] studied the incidence of malaria in two different zones of Punjab and introduce that PCR-based diagnostic is more precise and specific than the microscopy method. Steenkeste et al. [63] observed that the most effective approach for conducting extensive epidemiological investigation is the use of PCR-based diagnosis, epically in identifying mixed infections.